Apoptosis resistance plays a key role in malignant transformation and metastasis of the breast. Our goal is to understand mechanisms underlying apoptosis resistance in breast cancer by characterizing the role of integrin-extracellular matrix interactions in mammary epithlelial cell (MEC) survival. We found that laminin ligation of alpha6beta4 integrin and activation of RAC and NFkappaB are important for MEC survival, and that the efficiency of this pathway, irrespective of growth or malignancy status, is significantly enhanced when MECs assemble into polarized 3-dimensional (3D) tissue-like structures. We determined that apoptosis resistance in 3D tissue structures is associated with global repression of gene expression, decreased inducibility of NFkappaB inflammatory genes, altered AP-1 activity, reduced JNK activity and increased expression and activity of SMRT/N-CoR/HDAC. Because inhibiting SAPK induces SMRT/N-CoR/HDAC activity and protects MECs grown in 3D from apoptosis induction, and reducing SMRT/N-CoR expression or HDAC activity in 3D tissue structures permits death via exogenous apoptotic stimuli, we predict that alpha6beta4 integrin directs survival in MECs by facilitating activation of RAC and NFkappaB, but that the efficiency of this apoptosis resistance pathway is tempered by crosstalk between SAPK and SMRT/N-CoR/HDAC. The objective of the research outlined in this proposal is to define how alpha6beta4 integrin directs MEC survival through RAC and NFkappaB, and to explore the role of the SAPK pathway and SMRT/N-CoR activity as critical regulators of this pathway. We will achieve this goal by answering the following questions: 1. How does alpha6beta4 integrin activate NFkappaB and RAC? We will use mutant beta4 integrin expression constructs and MECs from beta4 integrin mutant mice, together with signaling intermediate constructs, NFkappaB functional assays and a PAK pulldown assay to map how beta4 integrin regulates NFkappaB and RAC, and biochemical manipulations with proteomics to characterize signaling candidates. 2. How does RAC activate NFkappaB to regulate MEC survival? We will use mutant RAC GTPases and signaling intermediates, and RNAi strategies as well as MECs isolated from a PAK null mouse to probe the role of and to identify candidate effectors of RAC linked to NFkappaB activation, and GST constructs and proteomics to map novel RAC cellular targets. 3. How does NFkappaB mediate MEC survival in 2D and 3D? We will use microarrays, CHIP, gel shift and fluorescence rescue assays to identify NFkappaB target genes and characterize transcriptional mechanisms, and expression and RNAi strategies to assess functional relevance to apoptosis resistance. 4. What is the role of SAPK-SMRT/N-CoR crosstalk in MEC survival? We will use mutant and wild type expression constructs, together with RNAi and biochemical assays to characterize the role of SAPK-SMRT/N-CoR crosstalk in MEC survival in culture. Our studies should facilitate the development of rational tumor therapies.